We have recently identified the PKI55 protein, coding for 55 amino acids, that is normally poorly translated in vivo and acts as a specific modulator of either cPKC-α and nPKC-δ isozymes. PKI55 remains relatively inactive until PKC attains an active conformation and reaches a critical concentration in the cell; it is not modified by the enzyme but irreversibly associates with its target, thus behaving as a suicidal inhibitor. The inhibition and degradation of over-activated PKC isozymes may prevent unfavorable changes in cellular phenotypes, resulting from PKC overexpression (Selvatici, J Mol Evol 2003 57:131). In the present work we compared the in vitro biochemical activity of the PKI55 protein, of its 39-amino acids N-terminal fragment (G39) and of its 16 amino acids C-terminal fragment (G16) on specific PKC recombinant isozymes, by measuring the initial rate of phosphate incorporation from 32P-ATP into saturating amounts of histone IIIS. PKI55 concentration-dependently inhibited PKC activity, provided that calcium ions were present in the assay medium (IC50=6μM). The inhibitory activity was retained by G16 (IC50=50μM), but not by G39. The active fragment G16 was tested in an in vitro model of brain ischemia: superfused guinea pig cerebral cortex slices, continuously electrically (10 Hz) stimulated, were exposed to 20 min of oxygen-glucose deprivation (OGD, Badini, Neurochem Int 1997 31:817), and then reperfused for 1 hour (REP). PKC activity was increased to 244±36% at the end of OGD, while acetylcholine (ACh) release, taken as an index of the neurosecretory function, was reduced to 35±2% of the controls. Following REP, a reduction in PKC activity to 54±7% was displayed, indicating a down regulation of previously activated PKC (Selvatici, J Neurosci Res 2003 71:64). ACh release only partially recovered (REP=69±6% of the controls), suggesting persistence of neuronal suffering. PKC activation during OGD was attenuated by 50µM G16 and the consequent down regulation following REP was prevented. Moreover, ACh release fully recovered to normal values (REP+G16=91±4% of the controls). These data suggest a neuroprotective action for G16, the active fragment of the endogenous PKC inhibitor PKI55.

We have recently identified the PKI55 protein, coding for 55 amino acids, that is normally poorly translated in vivo and acts as a specific modulator of either cPKC-α and nPKC-δ isozymes. PKI55 remains relatively inactive until PKC attains an active conformation and reaches a critical concentration in the cell; it is not modified by the enzyme but irreversibly associates with its target, thus behaving as a suicidal inhibitor. The inhibition and degradation of over-activated PKC isozymes may prevent unfavorable changes in cellular phenotypes, resulting from PKC overexpression (Selvatici, J Mol Evol 2003 57:131). In the present work we compared the in vitro biochemical activity of the PKI55 protein, of its 39-amino acids N-terminal fragment (G39) and of its 16 amino acids C-terminal fragment (G16) on specific PKC recombinant isozymes, by measuring the initial rate of phosphate incorporation from 32P-ATP into saturating amounts of histone IIIS. PKI55 concentration-dependently inhibited PKC activity, provided that calcium ions were present in the assay medium (IC50=6μM). The inhibitory activity was retained by G16 (IC50=50μM), but not by G39. The active fragment G16 was tested in an in vitro model of brain ischemia: superfused guinea pig cerebral cortex slices, continuously electrically (10 Hz) stimulated, were exposed to 20 min of oxygen-glucose deprivation (OGD, Badini, Neurochem Int 1997 31:817), and then reperfused for 1 hour (REP). PKC activity was increased to 244±36% at the end of OGD, while acetylcholine (ACh) release, taken as an index of the neurosecretory function, was reduced to 35±2% of the controls. Following REP, a reduction in PKC activity to 54±7% was displayed, indicating a down regulation of previously activated PKC (Selvatici, J Neurosci Res 2003 71:64). ACh release only partially recovered (REP=69±6% of the controls), suggesting persistence of neuronal suffering. PKC activation during OGD was attenuated by 50µM G16 and the consequent down regulation following REP was prevented. Moreover, ACh release fully recovered to normal values (REP+G16=91±4% of the controls). These data suggest a neuroprotective action for G16, the active fragment of the endogenous PKC inhibitor PKI55.